Exploiting PI3K/mTOR signaling to accelerate epithelial wound healing

The molecular circuitries controlling the process of skin wound healing have gained new significant insights in recent years. This knowledge is built on landmark studies on skin embryogenesis, maturation, and differentiation. Furthermore, the identification, characterization, and elucidation of the biological roles of adult skin epithelial stem cells and their influence in tissue homeostasis have provided the foundation for the overall understanding of the process of skin wound healing and tissue repair. Among numerous signaling pathways associated with epithelial functions, the PI3K/Akt/mTOR signaling route has gained substantial attention with the generation of animal models capable of dissecting individual components of the pathway, thereby providing a novel insight into the molecular framework underlying skin homeostasis and tissue regeneration. In this review, we focus on recent findings regarding the mechanisms involved in wound healing associated with the upregulation of the activity of the PI3K/Akt/mTOR circuitry. This review highlights critical findings on the molecular mechanisms controlling the activation of mTOR, a downstream component of the PI3K–PTEN pathway, which is directly involved in epithelial migration and proliferation. We discuss how this emerging information can be exploited for the development of novel pharmacological intervention strategies to accelerate the healing of critical size wounds.     

Transferability and Fine Mapping of Type 2 Diabetes Loci in African Americans: The Candidate Gene Association Resource Plus Study

Type 2 diabetes (T2D) disproportionally affects African Americans (AfA) but, to date, genetic variants identified from genome-wide association studies (GWAS) are primarily from European and Asian populations. We examined the single nucleotide polymorphism (SNP) and locus transferability of 40 reported T2D loci in six AfA GWAS consisting of 2,806 T2D case subjects with or without end-stage renal disease and 4,265 control subjects from the Candidate Gene Association Resource Plus Study. Our results revealed that seven index SNPs at the TCF7L2, KLF14, KCNQ1, ADCY5, CDKAL1, JAZF1, and GCKR loci were significantly associated with T2D (P < 0.05). The strongest association was observed at TCF7L2 rs7903146 (odds ratio [OR] 1.30; P = 6.86 × 10⁻⁸). Locus-wide analysis demonstrated significant associations (P_emp < 0.05) at regional best SNPs in the TCF7L2, KLF14, and HMGA2 loci as well as suggestive signals in KCNQ1 after correction for the effective number of SNPs at each locus. Of these loci, the regional best SNPs were in differential linkage disequilibrium (LD) with the index and adjacent SNPs. Our findings suggest that some loci discovered in prior reports affect T2D susceptibility in AfA with similar effect sizes. The reduced and differential LD pattern in AfA compared with European and Asian populations may facilitate fine mapping of causal variants at loci shared across populations.Type 2 diabetes (T2D) is a major public health problem affecting 25.8 million people in the U.S. (1). Marked racial differences in its prevalence have been observed, with African American (AfA) adults >40 years of age having nearly twofold higher prevalence than European Americans (27.1 and 15.5%, respectively) (2). In addition to socioeconomic and behavioral risk factors, genetic factors are likely contributors to T2D risk in AfA (3).Genome-wide association studies (GWAS) for T2D and related traits have successfully identified >50 loci with common genetic variants associated with T2D risk in primarily European-descent populations (4–14) and more recently in East and South Asians (15–21). The reported index single nucleotide polymorphisms (SNPs) at these loci have been replicated in multiple populations (22–24) but less successfully in AfA (25–27). Although differences in environment and lack of study power may partly account for the lack of transferability across ethnicities, differences in linkage disequilibrium (LD) patterns, effect sizes, and risk allele frequency also likely impact the replication of index SNPs. Although the long-range LD in European populations allows for the identification of T2D loci using less dense markers, causal variants are not distinguishable from other nearby SNPs in high LD. This issue prompts the need to examine T2D loci in other populations with different allelic and LD architecture, which may help fine mapping of the underlying functional variants (28).We performed a comprehensive evaluation of the LD region of T2D loci reported in European and Asian GWAS in a meta-analysis of six AfA GWAS. By testing the index and nearby SNPs, we evaluated the transferability of the previously reported loci for T2D association in AfA. We demonstrated that the reduced and differential LD structure in AfA facilitated fine mapping of regions potentially harboring causal variants at some T2D loci.     

On Efficient and Accurate Calculation of Significance P‐Values for Sequence Kernel Association Testing of Variant Set

The objective of this paper is to discuss and develop alternative computational methods to accurately and efficiently calculate significance P‐values for the commonly used sequence kernel association test (SKAT) and adaptive sum of SKAT and burden test (SKAT‐O) for variant set association. We show that the existing software can lead to either conservative or inflated type I errors. We develop alternative and efficient computational algorithms that quickly compute the SKAT P‐value and have well‐controlled type I errors. In addition, we derive an alternative and simplified formula for calculating the significance P‐value of SKAT‐O, which sheds light on the development of efficient and accurate numerical algorithms. We implement the proposed methods in the publicly available R package that can be readily used or adapted to large‐scale sequencing studies. Given that more and more large‐scale exome and whole genome sequencing or re‐sequencing studies are being conducted, the proposed methods are practically very important. We conduct extensive numerical studies to investigate the performance of the proposed methods. We further illustrate their usefulness with application to associations between rare exonic variants and fasting glucose levels in the Atherosclerosis Risk in Communities (ARIC) study.     

Monoclonal antibodies recognizing epitopes on the extracellular face and intracellular N-terminus of the human erythrocyte anion transporter (band 3) and their application to the analysis of South East Asian ovalocytes

This report describes the production and characterization of 13 rodent monoclonal antibodies to the human erythrocyte anion transport protein AE1 (syn. band 3). Eleven antibodies (4 murine and 7 rat) recognize epitopes dependent on the integrity of the third extracellular loop of the protein. Two antibodies (1 murine and 1 rat) recognize epitopes on the N-terminal cytoplasmic domain. Quantitative binding studies using radioiodinated IgG and Fab fragments of antibodies to extracellular epitopes on AE1 ranged from 77,000 to 313,000 (IgG) and from 241,000 to 772,000 (Fab) molecules bound at saturation. The results indicate that the epitopes recognized by different antibodies vary in their accessibility and suggest that there is heterogeneity in the organization of individual AE1 molecules in the red blood cell membrane. Quantitative binding studies on South East Asian ovalocytes using several antibodies to AE1 and an anti-Wrb show a marked reduction in the number of antibody molecules bound at saturation. These results are consistent with the existence of highly cooperative interactions between transmembrane domains of AE1 in normal erythrocytes and the disruption of these interactions in the variant AE1 found in South East Asian ovalocytes.     

Maternal administration of granulocyte colony-stimulating factor improves neonatal rat survival after a lethal group B streptococcal infection

Maternally administered recombinant human granulocyte colony- stimulating factor (rhG-CSF) has been shown to cross the placenta and induce a peripheral neutrophilia and increases in the marrow and spleen neutrophil storage pools in fetal and newborn rats. In the present study, we have used this model system to investigate the efficacy of prenatally administered rhG-CSF on neonatal defense to a lethal challenge with Group B-beta hemolytic Streptococcus (GBS). Pregnant rats were injected with rhG-CSF twice daily beginning 6 days before parturition. At birth, all pups were infected with a dose of GBS that is lethal for 90% of infected pups (LD90). Survival was monitored daily for 5 days. Survival of infected pups from saline-treated mothers beyond 60 hours after infection was 10%. No difference in survival was observed among pups from mothers treated 2 and 4 days before parturition. In contrast, we determined that survival was 82.5% among infected pups from mothers treated for 6 days before parturition with rhG-CSF. Our results demonstrate that maternal administration of rhG- CSF augments neonatal defenses against a lethal bacterial challenge.     

Granulocyte-macrophage colony-stimulating factor augmentation of T-cell receptor-dependent and T-cell receptor-independent thymocyte proliferation

The effects of granulocyte-macrophage colony-stimulating factor (GM- CSF) are not confined to cells of the myeloid lineage. GM-CSF has been shown to have effects on mature T cells and both mature and immature T- cell lines. We therefore examined the GM-CSF responsiveness of murine thymocytes to investigate whether GM-CSF also affected normal immature T lymphocytes. The studies presented here indicate that GM-CSF augments accessory cell (AC)-dependent T-cell receptor (TCR)-mediated proliferation of unseparated thymocyte populations. To identify the GM- CSF responsive cell type, thymic AC and T cells were examined for GM- CSF responsiveness. We found that GM-CSF augmentation of TCR-induced thymocyte proliferation appears to be mediated via augmentation of AC function, and not via direct effects on mature single-positive (SP) thymocytes. Enriched double-negative (DN) thymocytes were also tested for GM-CSF responsiveness. GM-CSF induced the proliferation of adult and fetal DN thymocytes in an AC-independent and TCR-independent single- cell assay. Thus, in contrast to the SP thymocytes, a DN thymocyte population was directly responsive to GM-CSF. GM-CSF therefore may play a direct role in the expansion of DN thymocytes and an indirect role in the expansion of SP thymocytes.     

Hematopoietic growth factors for graft failure after bone marrow transplantation: a randomized trial of granulocyte-macrophage colony- stimulating factor (GM-CSF) versus sequential GM-CSF plus granulocyte- CSF

Delay in hematologic recovery after bone marrow transplantation (BMT) can extend and amplify the risks of infection and hemorrhage, compromise patients' survival, and increase the duration and cost of hospitalization. Because current studies suggest that granulocyte- macrophage (GM) colony-stimulating factor (CSF) may potentiate the sensitivity of hematopoietic progenitor cells to G-CSF, we performed a prospective, randomized trial comparing GM-CSF (250 micrograms/m2/d x 14 days) versus sequential GM-CSF x 7 days followed by G-CSF (5 micrograms/kg/d x 7 days) as treatment for primary or secondary graft failure after BMT. Eligibility criteria included failure to achieve a white blood cell (WBC) count > or = 100/microL by day +21 or > or = 300/microL by day +28, no absolute neutrophil count (ANC) > or = 200/microL by day +28, or secondary sustained neutropenia after initial engraftment. Forty-seven patients were enrolled: 23 received GM-CSF (10 unrelated, 8 related allogeneic, and 5 autologous), and 24 received GM- CSF followed by G-CSF (12 unrelated, 7 related allogeneic, and 5 autologous). For patients receiving GM-CSF alone, neutrophil recovery (ANC > or = 500/microL) occurred between 2 and 61 days (median, 8 days) after therapy, while those receiving GM-CSF+G-CSF recovered at a similar rate of 1 to 36 days (median, 6 days; P = .39). Recovery to red blood cell (RBC) transfusion independence was slow, occurring 6 to 250 days (median, 35 days) after enrollment with no significant difference between the two treatment groups (GM-CSF: median, 30 days; GM-CSF+G- CSF; median, 42 days; P = .24). Similarly, platelet transfusion independence was delayed until 4 to 249 days (median, 32 days) after enrollment, with no difference between the two treatment groups (GM- CSF: median, 28 days; GM-CSF+G-CSF: median, 42 days; P = .38). Recovery times were not different between patients with unrelated donors and those with related donors or autologous transplant recipients. Survival at 100 days after enrollment was superior after treatment with GM-CSF alone. Only 1 of 23 patients treated with GM-CSF died versus 7 of 24 treated with GM-CSF+G-CSF who died 16 to 84 days (median, 38 days) after enrollment, yielding Kaplan-Meier 100-day survival estimates of 96% +/- 8% for GM-CSF versus 71% +/- 18% for GM-CSF+G-CSF (P = .026). These data suggest that sequential growth factor therapy with GM-CSF followed by G-CSF offers no advantage over GM-CSF alone in accelerating trilineage hematopoiesis or preventing lethal complications in patients with poor graft function after BMT.(ABSTRACT TRUNCATED AT 400 WORDS)     

Estimation of Geographic Variation in Human Papillomavirus Vaccine Uptake in Men and Women: An Online Survey Using Facebook Recruitment

Background

Federally funded surveys of human papillomavirus (HPV) vaccine uptake are important for pinpointing geographically based health disparities. Although national and state level data are available, local (ie, county and postal code level) data are not due to small sample sizes, confidentiality concerns, and cost. Local level HPV vaccine uptake data may be feasible to obtain by targeting specific geographic areas through social media advertising and recruitment strategies, in combination with online surveys.

Objective

Our goal was to use Facebook-based recruitment and online surveys to estimate local variation in HPV vaccine uptake among young men and women in Minnesota.

Methods

From November 2012 to January 2013, men and women were recruited via a targeted Facebook advertisement campaign to complete an online survey about HPV vaccination practices. The Facebook advertisements were targeted to recruit men and women by location (25 mile radius of Minneapolis, Minnesota, United States), age (18-30 years), and language (English).

Results

Of the 2079 men and women who responded to the Facebook advertisements and visited the study website, 1003 (48.2%) enrolled in the study and completed the survey. The average advertising cost per completed survey was US $1.36. Among those who reported their postal code, 90.6% (881/972) of the participants lived within the previously defined geographic study area. Receipt of 1 dose or more of HPV vaccine was reported by 65.6% women (351/535), and 13.0% (45/347) of men. These results differ from previously reported Minnesota state level estimates (53.8% for young women and 20.8% for young men) and from national estimates (34.5% for women and 2.3% for men).

Conclusions

This study shows that recruiting a representative sample of young men and women based on county and postal code location to complete a survey on HPV vaccination uptake via the Internet is a cost-effective and feasible strategy. This study also highlights the need for local estimates to assess the variation in HPV vaccine uptake, as these estimates differ considerably from those obtained using survey data that are aggregated to the state or federal level.